SHERLOCK, a novel CRISPR-Cas13a-based assay for detection of infectious bursal disease virus

Abstract

Infectious bursal disease (IBD) is an extremely contagious viral infection that primarily affects young chicks, leading to significant economic losses in the poultry industry. The disease is caused by a double-stranded RNA virus of the genus Avibirnavirus, family Birnaviridae, namely, the infectious bursal disease virus (IBDV). Unfortunately, current methods for detecting IBDV lack adequate sensitivity. Accordingly, the advantages of the Specific High Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) assay were employed to develop an ultrasensitive assay (IBD-SHERLOCK assay) for the detection of IBDV in clinical chicken tissues. The assay comprises two steps: isothermal preamplification of the target RNA through reverse transcription recombinase polymerase amplification (RT-RPA) and a subsequent detection step, which is based on the CRISPR-Cas13a system. The integration of lateral flow (LFD) visual detection of the IBD-SHERLOCK products strengthens the feasibility of the assay for use as a point-of-care test in chicken farms. Compared with RT–qPCR, this method exhibited ultra-analytical and clinical sensitivity. The assay has a lower detection limit of 5 aM, which is equivalent to three IBDV-RNA molecules. The assay demonstrated the ability to detect IBDV-RNA in 70 clinical field samples, 15 of which tested negative by RT–qPCR. This evidence highlights its superior sensitivity and potential for early detection of IBDV in chicken tissues. This study effectively established and verified a CRISPR-based diagnostic test for the early detection of IBDV in clinical chicken tissues, demonstrating remarkable specificity and sensitivity. The IBD-SHERLOCK assay can be used as a highly sensitive point-of-care diagnostic tool in chicken farms.